Genomic Dissection of Bacterial Pathogens
To better elucidate mechanisms of pathogenicity, our laboratory is developing powerful, high-throughput models to study the infectious process and combining them with the power of bacterial genetics and genomics. Our lab has studied a number of microbial pathogens over the past several years, including species of Salmonella and Enterococcus, Staphylococcus aureus, Cryptococcus neoformans, and Fusarium oxysporum. However, the first human pathogen studied in the lab, and the best characterized, is the opportunistic pathogen, Pseudomonas aeruginosa.
Pseudomonas aeruginosa, a ubiquitous Gram-negative bacterium isolated from soil, water, and plants, is an opportunistic pathogen that infects cystic fibrosis patients, patients with thermal burns and patients who are immunodeficient or otherwise compromised. A number of years ago, we discovered that a clinical isolate, called PA14, could infect both plants and animals, and that the ability to cause disease in plants required virulence factors known to be important for pathogenicity in mammalian hosts, arguing for a conservation of mechanism in this process. Using PA14, we have developed a multi-host pathogenesis system in which the bacterium is used to infect nematodes and insects in addition to traditional cell cultures and murine hosts. These simpler model hosts are genetically tractable, reproduce more rapidly, and are smaller and cheaper to raise than their mammalian counterparts, allowing for large-scale, high-throughput assays of virulence that are impractical or unethical to perform using mice.
The Ausubel lab is currently developing PA14 tools to better characterize its behavior during infection. The most common laboratory strain of P. aeruginosa is PAO1, which was sequenced in 2000. However, this strain is generally less virulent than PA14 in a number of hosts. We have therefore sequenced the genome of PA14 to begin to understand the strain differences that account for its enhanced pathogenicity. In conjunction with the sequencing of PA14, we have created a genome-wide collection of defined, non-redundant mutations (http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi). Thirty thousand independent transposon insertion mutations were generated and each isolate was sequenced to determine the site of the lesion. From this master set, we then selected 5850 mutants corresponding to 75% of the total and approximately 80% of the non-essential PA14 ORFs. This non-redundant unigene library allows for rapid, near-saturated screens of PA14 in a number of assays, examining virulence or any other aspect of Pseudomonas biology. We used this non-redundant transposon mutant library to carry out a genome-wide screen for attenuation of PA14 virulence in C. elegans. We defined a functionally diverse 180 mutant set (representing 170 unique genes) necessary for normal levels of virulence that included both known and novel virulence factors. We examined the collection of genes required for normal levels of PA14 virulence with respect to occurrence in P. aeruginosa strain-specific genomic regions, location on putative and known genomic islands, and phylogenetic distribution across prokaryotes. Genes predominantly contributing to virulence in C. elegans showed no bias for either strain-specific regions of the P. aeruginosa genome nor putatively horizontally transferred genomic islands. Instead, within the collection of virulence-related PA14 genes, there was an over-representation of genes with a broad phylogenetic distribution that also occur with high frequency in many prokaryotic clades, suggesting that in aggregate the genes required for PA14 virulence in C. elegans are biased towards evolutionarily conserved genes.